DNA double strand breaks (DSBs) are cytotoxic lesions that occur spontaneously during normal cell metabolism or by treatment of cells with DNA-damaging agents. If unrepaired or repaired inappropriately, DSBs can lead to mutagenic events, such as chromosome loss, deletions, duplications or translocations, events that can lead to carcinogenesis. The homology-dependent repair of DSBs usually occurs by a conservative gene conversion mechanism, preventing extensive loss of heterozygosity (LOH) or chromosome rearrangements. Breaks that present only one end for repair, for example at eroded uncapped telomeres or when homology is limited to one side of the DSB, are thought to repair by strand invasion into a homologous duplex DNA followed by replication to the chromosome end (break-induced replication, BIR). As BIR from one of the two ends of a DSB would result in extensive LOH it suggests BIR is suppressed when DSBs have two ends in order for repair to occur by a more conservative HR mechanism. Furthermore, our studies have shown that the replication intermediate formed during BIR is unstable and the invading end can switch to a different template resulting in a translocation. BIR and a related mechanism, fork stalling and template switching (FoSTeS), are thought to be responsible for many of the genome rearrangements that give rise to non-reciprocal translocations and copy number variation associated with human disease. The goals of this proposal are to understand the mechanisms of BIR and how cells switch from gene conversion to the BIR mode of repair. The specific aims are: (1) To use a new genetic assay that detects template switching to identify the genes that regulate this process. (2) To determine whether there is a bias in the use of ectopic templates for non-reciprocal translocations that arise during BIR. (3) Physical methods will be used to determine whether DNA synthesis associated with BIR is conservative or non-conservative, the rate of fork movement will be determined by DNA combing, and the role of different DNA polymerases and the Mcm2-7 replicative helicase in the initiation and completion of BIR will be determined. PUBLIC HEALTH RELEVANCE: The repair of DNA double-strand breaks by break-induced replication (BIR) can lead to several types of chromosome rearrangements that are associated with human disease. BIR is also involved in maintaining telomere length in the absence of telomerase and this process is activated in some human tumors. In this proposal, genetic and physical approaches will be used to identify the genes that regulate chromosome rearrangements during BIR, and to define the mechanism for DNA synthesis used in BIR.